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5 minutes

The authors thank Adriana Montserrat for administrative and purchasing management. A. Ronceret acknowledges grants from UNAM-PAPIIT and SECIHTI, and thanks the Institute of Biotechnology (IBt-UNAM) for initial and annual funding. P. Bola&#241;os thanks the Vice-Rectorate for Research at the University of Costa Rica for ongoing support.

CAUTION: Handle formaldehyde and acrylamide using appropriate personal protective equipment and a chemical hood; both are toxic (acrylamide is also neurotoxic before polymerization). All the materials used in the study are listed in the Table of Materials.
1. Fixation of maize anthers to preserve the intact structure of meiocytes
<ol>
	<li>Use maize plants at the V13 stage with inflorescences still growing inside the stem. During the week of meiotic anther fixation, wear gloves and gently press the enlarged stem at the bump to determine if it&#8217;s soft enough to cut open. 
	NOTE: This protocol for fixation and immunolocalization may be used for both maize and Arabidopsis thaliana meiotic flower buds (less than 400 &#181;m). But in the case of maize, depending on the variety used and the time of the year (there is quicker growth in spring), it takes around 8 weeks from planting for reference inbred line B73, but only 6 weeks for the early flowering inbred line A344.</li>
	<li>Preparation of buffers. 
	NOTE: The day before chemical fixation, prepare the following working solutions, do it carefully, and label all bottles properly.
	<ol>
		<li>Prepare 100 mL of 10x Buffer A (BA) salts, by adding 4.53 g PIPES (Piperazine-N, N&#8242;-bis(2-ethanesulfonic acid) at a final concentration of 150 mM), 5.96 g KCl (final concentration 800 mM), 1.17 g NaCl (final concentration 200 mM), 0.74 g EDTA (final concentration 20 mM), 0.19 g EGTA (final concentration 5 mM) and 90 mL H2O, first adjust pH to 6.8&#8211;7 with KOH, then adjust the volume to 100 mL.</li>
		<li>Finally, filter sterilize using a disposable Rapid-Flow sterile filter unit with PES of 0.45 &#181;m pore (keep at 4 &#176;C but do not freeze; you may store it for several months).</li>
		<li>Prepare: 
		-50 mM PIPES (pH adjusted to 7 with KOH). 
		- stocks of spermine at a concentration of 0.4 M (previously dissolved in 50 mM PIPES, adjust pH to 7 with KOH, keep aliquots of 50 &#956;L at -20 &#176;C). 
		- stocks of spermidine at a concentration of 0.4 M (dissolved in 50 mM PIPES, with pH adjusted to 7 with KOH, keep frozen aliquots of 125 &#956;L at -20&#176;C). 
		- stocks of Dithiothreitol (DTT) 1 M (dissolved in 0.01 M sodium acetate, to pH 5.2, keep frozen aliquots of 100 &#956;L at -20 &#176;C). 
		- sorbitol 2 M, kept at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 50 mL of 2x Buffer A (BA), on ice, using 10 mL of 10x Buffer A salts (kept at 4 &#176;C), 50 &#956;L of spermine (0.4 M in 50 mM PIPES, with pH adjusted to 7 with KOH), 125 &#956;L of spermidine (0.4 M in 50 mM PIPES, with pH adjusted to 7 with KOH), 100 &#956;L of DTT (1 M in 0.01 M sodium acetate, pH 5.2) vortex vigorously to dissolve; and do not heat.
	<ol>
		<li>Then, add 16 mL of 2 M sorbitol. Adjust the final volume to 50 mL with sterile water. Filter-sterilize using a disposable Rapid-Flow sterile filter and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare a humid chamber made of 5&#8211;10 humidified paper towels placed in a plastic box. Verify that the lid seals well. Eliminate any excess water.</li>
	<li>Then, place in a tray the following items: the humid chamber, a razor blade, fine forceps (previously sterilized with 70% ethanol), a small bottle of sterile water, and masking tape. Bring the tray and its contents to the maize plants checked at step 1.1.</li>
	<li>Use a razor blade to cut open the bumpy stem at the level of the inflorescence. The main tassel branches, as well as the lateral branches of the inflorescence, can be used for harvesting meiotic florets. 
	NOTE: These tassel branches contain the florets of interest to collect meiotic anthers. Each floret contains six anthers, and the 3 upper, larger ones are typically used for analysis. The approximate size and color of the anthers correlate with each meiotic stage. Almost transparent anthers around 1 mm in length are at the leptotene stage, translucent anthers at 1.5 mm in length are at the zygotene stage, whitish anthers at 2 mm in length are at the pachytene stage, and yellowish anthers at &#62; 2 mm in length may contain meiocytes at diplotene, tetrads, and pollen grains that finish gametogenesis. Note that another length-stage correlation can vary among maize genetic backgrounds, and that environmental factors (e.g., high temperature) can accelerate meiotic progression and alter this relationship. It is recommended that researchers validate anther size-stage correlations for each specific maize genotype and growth conditions (via acetocarmine squashing, cf 1.6).</li>
	<li>Use fine forceps to dissect and carefully collect the lateral branches of the inflorescences and wrap them inside the paper towel of the humid chamber. 
	NOTE: With some practice, only a short linear 3&#8211;4 cm incision in the stem is needed to extract the branches of the meiotic tassel. You may close this incision by wrapping it three to four times with standard masking tape, which allows you to later harvest the same plant using the remaining undissected inflorescence. Tissue will keep growing and later may be used for pollination.</li>
	<li>Keep the chamber at room temperature. Use only those branches of the inflorescence that contain florets that carry anthers at the meiotic stage. Make sure to cover the branches with half of the humid towels, but do not immerse them in water.</li>
	<li>Humidify again if the towels dry out. Close the box until dissection of the anthers, preferably until 30 min before the start of anther collection.</li>
	<li>Using a binocular stereomicroscope with a calibrated scale, collect fresh anthers from florets into 2 mL of 1x BA in a 35 mm Petri dish, ensuring accurate identification of meiotic stages using the proxy of anther size and color. 
	NOTE: To accurately determine the meiocyte stage in sampled anthers, one of three synchronized anthers per floret is quickly analyzed using 3:1 ethanol-acetic acid fixation, acetocarmine staining, and traditional squash microscopy13.</li>
	<li>Prepare the 4% formaldehyde fixative in 1x BA and 0.0025% Tween-20 
	NOTE: For 3 mL, add 750 &#956;L formaldehyde 16%, 750 &#956;L H2O, 1.5 mL 2x BA, and 7.5 &#956;L Tween-20 at 1%.</li>
	<li>Remove the initial BA buffer and add the 3 mL of this fresh fixative solution to the staged anthers and have them fixated for 45 min inside a new 35 mm Petri dish, sealed by parafilm to prevent formaldehyde evaporation, then place it on a larger Petri dish placed on a rotary shaker (set at 40&#8211;60 rpm, or 0.2 x g). Do it at room temperature. Anthers should be swirling slightly for good fixation. They usually float on the surface.</li>
	<li>Rinse with 3 mL of 1x BA, then transfer the sample to a small Petri dish and place it on a rotary shaker (40&#8211;60 rpm) at room temperature for 45 min.</li>
	<li>Using clean forceps, transfer the anthers to a small 35 mm Petri dish containing fresh 3 mL of 1X BA. Seal the Petri dish with parafilm tightly. Then store the small Petri dish inside a Petri dish, covering it with aluminum foil to keep it in the dark, and place it at 4 &#176;C until use. You may store it for several months if needed.</li>
</ol>
2. Embedding maize meiocytes within polyacrylamide sandwich pads
DAY 1: 3D slides preparation
<ol>
	<li>Prepare 2 mL of acrylamide solution by mixing equal volumes (1:1) of acrylamide and 2x BA. Before adding sodium sulfite (NaS) and ammonium persulfate (APS), degas the acrylamide solution under vacuum for 5&#8211;10 min.</li>
	<li>Aliquot the acrylamide and 2x BA 1:1 ratio mixes by pipetting 100 &#181;L into 1.5 mL tubes. 
	The number of tubes you prepare depends on the number of slides you will prepare.</li>
	<li>Make 1 mL 1x BA.</li>
	<li>Place 2 cm x 2 cm pieces of parafilm over a Petri dish, then place them under a dissecting microscope. Pipette 50 &#181;L of BA solution with anthers onto parafilm. Trim the pipette tip with scissors for easier anther delivery.
	<ol>
		<li>Pipette 25 &#181;L of BA solution onto parafilm elsewhere.</li>
	</ol>
	</li>
	<li>Using forceps to hold anthers, cut the tip of the anthers with a scalpel, and gently squeeze the meiocytes out. With a pipette, transfer the meiocytes gently to the 25 &#181;L BA drop and gently mix.</li>
	<li>Pipette 10 &#181;L from the meiocyte mix onto a separate cover slip.</li>
	<li>Rapidly, pipette 5 &#181;L of 20% (0.2g/ 1mL) NaS and 5 &#181;L 20% (0.2g/ 1mL) APS into a 1.5 mL tube with Acrylamide/2x Buffer A.</li>
	<li>Rapidly, pipette 5 &#181;L of the above mix onto a coverslip and mix with meiocytes. Place a poly-L-lysine-capped coverslip on top, but rotate it 45 degrees. Using only one poly-L-lysine coverslip (either commercially obtained or made by immersing coverslips in a poly-L-lysine solution in a Petri dish for a few minutes and letting them dry, on the edge of a new, clean Petri dish) helps the release of a complete pad after polymerization. The coverslip is quickly lifted from the solution and dried before use. For super-resolution microscopy, high-performance coverslips with standardized thickness (&#8804;170 nm) must be used14.</li>
	<li>Please do not touch the coverslips for 30&#8211;50 min to allow full polymerization of the sample at room temperature (A quicker polymerization process reduces the drying period of the pad, and the resulting quality is less than optimal).</li>
	<li>Repeat steps &#8216;2.6&#8211;2.9&#8217; for each slide you wish to prepare.</li>
	<li>Separate all coverslips carefully with a razor blade. Place all coverslips, pad-side up, on a polyacrylamide pad (or most of the pad), in a 6-well plate.</li>
	<li>Wash each coverslip with a pad with 2 mL of 1x phosphate-buffered saline (PBS) for 10 mins. Repeat this wash once, for a total washing time of 20 min at room temperature.</li>
	<li>Prepare 50 mL by mixing 49.4 mL PBS 1x, 100 &#181;L EDTA 0.5 M, and 500 &#181;L Triton.</li>
	<li>Rinse each coverslip with a pad in 1 mL of this 1x PBS supplemented with 1 mM EDTA and 1% Triton for 2 h at room temperature.</li>
	<li>Prepare a fresh batch of blocking buffer containing 1x PBS, 1 mM EDTA, 0.1% Tween-20, and 3% BSA (1 mL per slide). For 50 mL of blocking buffer, mix 44 mL of PBS 1x, 100 &#181;L of EDTA 0.5 M, 5 mL of 1% Tween-20 solution, and 1.5 g of BSA. BSA is preferred over donkey serum as a blocking agent because it helps minimize non-specific signals when using multiple primary and secondary antibodies.</li>
</ol>
3. Immunolocalizations
<ol>
	<li>Prepare primary antibody (or antibodies) at the wanted dilution using blocking buffer. Combine primary antibodies based on their ability to produce distinct signals when used together. They should come from different organisms, such as rabbit, rat, guinea pig, or chicken, that can be recognized by specific fluorescently labelled secondary antibodies. The use of donkey or goat antibodies is avoided because they are intended for use as secondary antibodies. We recommend using a single species of secondary antibody (e.g., goat in the experiment shown in Figure 1).</li>
	<li>Add 50 &#181;L of this diluted primary antibody onto the center of each coverslip with a pad. 
	Add water between wells to slow down evaporation. Put the plate in a humid chamber (a plastic box with a lid). Leave overnight.</li>
</ol>
DAY 2: Washing
<ol start="3">
	<li>Prepare 500 mL of Wash Buffer (1x PBS + 0.1% Tween-20 + 1 mM EDTA).</li>
	<li>Remove the overnight solution from the wells by aspiration with your pipette.</li>
	<li>Quickly wash each coverslip with a pad twice with 1 mL of wash buffer, then remove it from the wells.</li>
	<li>Pipette another 1 mL/ coverslip with pad and leave sitting for 1 h at room temperature. 
	Repeat 7 more times. (8 h total)</li>
	<li>After the final wash, add water between wells, cover the plate, and leave it overnight in a humid chamber.</li>
</ol>
DAY 3: Secondary antibody, washes, and mounting slides
<ol start="8">
	<li>Prepare the secondary antibodies by diluting each at a ratio of 1:50 in blocking buffer. 
	Aspirate the wash solution. Dispense 50 &#181;L of the diluted secondary antibody onto the center of each pad using a pipette. 
	NOTE: Every secondary antibody should be used in association with distinct fluorescent dyes with non-overlapping emission wavelengths. For example, one secondary antibody should be labelled with AlexaFluor488 (green), the next one with AlexaFluor555 (orange), and the third with AlexaFluor647 (far red), which can be combined with DAPI (excitation at 461 nM, blue). Other fluorescent dyes, such as DyLight (by Dyomics GmbH), are also usable. In addition, all these secondary antibodies should not cross-react with other species used in the mix of primary antibodies. For example, the anti-rabbit secondary antibody should be cross-adapted to be selective for rabbit antibodies and not recognize either rat or guinea pig antibodies. The same is true for all secondary antibodies. A well-designed experiment should be used to validate all these requirements simultaneously when performing co-immunolocalizations.</li>
	<li>Add water between the wells, place a plate in the humid chamber, and leave for 2 h at room temperature.</li>
	<li>Aspirate the solution from the wells.</li>
	<li>Perform four washes using 1 mL per well, 1 h each at room temperature (4 h total).</li>
	<li>Wash each well with 1 mL of 1x PBS for 10 min at room temperature and repeat this process two more times. (30 min total)</li>
	<li>Dilute DAPI in 1x PBS.</li>
	<li>Stain with a final concentration of 1 &#181;g/mL DAPI (DAPI powder dissolved in water) for 30 min at room temperature, and place 500 &#181;L/well.</li>
	<li>Remove ProLong&#8482; Gold antifade from the 4 &#176;C freezer to allow it to thaw and reach room temperature.</li>
	<li>Wash slides in 1x PBS for 30 min at room temperature, with 1 mL/well. Do it twice again. (1 h 30 min total)</li>
	<li>Remove the last wash.</li>
	<li>Then add two drops of ProLong&#8482; Gold antifade in the middle of each pad.</li>
	<li>Mount cover slips on each slide, label every slide, lift the cover slips from the well using fine forceps, and slowly place them on the slide. Using fine forceps, slowly lower a second coverslip onto the first. Using a vacuum, remove excess glycerol from the edges of coverslips. Seal edges of coverslips with two coats of nail polish.</li>
	<li>If slides are not immediately examined under a fluorescent microscope. Store them in a -20 &#176;C freezer, protected from light, until observation. Later on, let slides sit at room temperature for 1 h in a slide box, horizontally and in the dark, to prevent condensation.</li>
</ol>
DAY 4: Microscopic observations
<ol start="21">
	<li>Observes the slide under the right excitation wavelengths and filters. Standard confocal microscopy is sufficient, but it is advisable to use a microscope with a 60x objective to enable three-dimensional imaging of the entire cell using a z-stack every 0,2 &#181;m, with sequential image acquisitions and deconvolution processing using the Applied Precision SoftWoRx&#174; software.</li>
</ol>

The authors declare no competing interests.

This protocol offers a key advantage over others by preserving essential three-dimensional data on meiotic chromosomal protein loading. These features cannot be preserved using regular spreading techniques1,2. Nonetheless, this protocol has two limitations: it is slow and technically demanding compared to regular spreading techniques performed within one day. In contrast, our protocol requires four days of work, mainly due to extensive washing steps and overnight...

Meiosis is a key process that regulates the transmission of traits in model plants and crops; thus, simple microscopy methods that allow consistent and faithful visualization of protein localization facilitate the understanding of how these traits could be transmitted more efficiently. Unfortunately, the visualization of the localization of proteins inside meiocytes has varied a lot depending on the technique used1,2. Thus, the aim of the protocol is to provide a straightforward, reproducible method that eliminates artifacts during the observation of meiocytes. For instance, it has long been acknowledged that the observation of meiotic chromosomes is a delicate process that is facilitated by crushing the cells by either simple squashing or by even more disruptive spreading techniques3,4,5. In Arabidopsis thaliana, researchers commonly employ spreading methods that utilize Carnoy&#8217;s fixative (a 3:1 mixture of ethanol and acetic acid) along with various detergents such as Lipsol, Tween-20, and TritonX-100, as well as enzyme mixtures containing cellulase, pectinase, and cytohelicases, to break apart cells and allow for effective immunolocalization on its small chromosomes1,2. These techniques transform a 3D chromosome conformation into a 2D one, enabling quick observation of the entirety of artificially dispersed chromosomes in a single image. Certain features of chromosomes that endure these harsh treatments may be apparent; however, they often fail to represent the typical behavior of cells and chromosomes in vivo6. Quick and very broad detection of up to two antibodies is possible with 2D spreading techniques in various plant species including maize7,8,9,10,11.
Nonetheless, despite the convenience of legacy 2D techniques, it is now acknowledged that certain phenomena are sensitive to disruptive methods and cannot be observed if the cell, nucleus, or chromosomes are damaged during spreading or squashing. For instance, the movement of the RAD50 protein from the cytoplasm to the nucleus during the leptotene stage, or during centromere coupling (leptonema/zygonema), has been detected only in intact 3D meiocytes12. Here, readers are offered a detailed 4-day 3D protocol that uses anthers fixed in 4% paraformaldehyde to immunolocalize three key proteins in maize male meiocytes, thereby preserving their structural integrity. Compared to a similar maize 3D immunolocalization methodology8, we have added practical notes and troubleshooting tips for researchers. This protocol will benefit the work of plant biologists and plant breeders interested in studying various meiotic processes, such as the control of meiotic recombination by the synaptonemal complex.

<table><tbody><tr><td>1.5 mL microtubes Axygen</td><td>Corning</td><td>MCT-150-C</td><td></td></tr><tr><td>15 mL Falcon tubes</td><td>Thermo Fisher Scientific</td><td>339651</td><td></td></tr><tr><td>50 mL Falcon tubes</td><td>Thermo Fisher Scientific</td><td>339652</td><td></td></tr><tr><td>6 well plate Falcon polystyrene microplate</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;08-772-1B</td><td></td></tr><tr><td>Acetic acid, 60%&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A38-212</td><td></td></tr><tr><td>Acrylamide 30%&amp;nbsp; and bis-acrylamide solution, 29:1</td><td>Biorad</td><td>1610157</td><td></td></tr><tr><td>Aluminum fold</td><td>Reynolds&amp;nbsp;</td><td>U07211R0</td><td></td></tr><tr><td>Amonium Persulfate (APS)</td><td>Merck</td><td>A3678</td><td></td></tr><tr><td>Binocular stereomicroscope</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)&amp;nbsp;</td><td>Merck</td><td>&amp;nbsp;A9418</td><td></td></tr><tr><td>DAPI</td><td>Merck</td><td>D9542</td><td></td></tr><tr><td>DelltaVision microscope</td><td>Olympus</td><td>NA</td><td></td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Disposable Filter Units with PES filters of 0.45 &amp;mu;m pore</td><td>Thermo Fisher Scientific/Nalgene</td><td>09-740-63B</td><td></td></tr><tr><td>Fine forceps FST Dumont SS</td><td>Fine Science Tools&amp;nbsp;</td><td>11200-33</td><td></td></tr><tr><td>goat (IgG H+L) highly cross-adsorbed&amp;nbsp; anti-rat labelled by AlexaFluor647</td><td>Invitrogen</td><td>A-21247</td><td></td></tr><tr><td>goat (IgG H+L) highly cross-adsorbed anti-rabbit labelled by AlexaFluor568</td><td>Invitrogen</td><td>A-21428</td><td></td></tr><tr><td>goat (IgG H+L) highly cross-adsorbed anti-guinea pig labelled by AlexaFluor488&amp;nbsp;</td><td>Invitrogen</td><td>A-11073</td><td></td></tr><tr><td>Gloves</td><td>Merck</td><td>Z412392</td><td></td></tr><tr><td>Growing chambers / greenhouse / or field</td><td>NA</td><td>NA</td><td></td></tr><tr><td>DTT</td><td>Merck</td><td>D0632</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Masking tape 3M</td><td>Fisher Scientific</td><td>19-047-259</td><td></td></tr><tr><td>Maize seeds</td><td>NA</td><td></td><td></td></tr><tr><td>Microscope 22 x 22 mm-1,5 glass coverslips&amp;nbsp;</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Microscope 22 x 22 mm-H coverslips&amp;nbsp;</td><td>Deckgl&amp;auml;ser, Zeiss</td><td>474030-9020-000</td><td></td></tr><tr><td>Microscopic glass slides</td><td>Fisher Scientific</td><td>22-230-900</td><td></td></tr><tr><td>Microscope slide box</td><td>Fisher Scientific,</td><td>03-448-9</td><td></td></tr><tr><td>Micropipets P20, P200, P1000</td><td>Merck</td><td>CLS4069</td><td></td></tr><tr><td>NaCl</td><td>Merck</td><td>S7653</td><td></td></tr><tr><td>Nail polish</td><td>Electron Microscopy Science</td><td>72180</td><td></td></tr><tr><td>Paper towels</td><td>Merck</td><td>Z188956</td><td></td></tr><tr><td>Parafilm</td><td>Merck</td><td>P7668</td><td></td></tr><tr><td>Paraformaldehyde 16% solution&amp;nbsp;</td><td>Electron Microscopy Grade</td><td>&amp;nbsp;15710</td><td></td></tr><tr><td>PBS</td><td>Merck</td><td>P4474</td><td></td></tr><tr><td>Plastic box with lid</td><td>Tupperware</td><td>NA</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Merck</td><td>P4707</td><td></td></tr><tr><td>Primary antibodies (rat anti-ZmAFD1, rabbit anti-ZmASY1, guinea-pig anti ZmZYP1)</td><td>NA</td><td></td><td></td></tr><tr><td>Prolong Antifading</td><td>Molecular Probes</td><td>P36934</td><td></td></tr><tr><td>Petri dish (D x H: 35 mm x 10 mm</td><td>Corning</td><td>351008</td><td></td></tr><tr><td>Petri dish regular size</td><td>Merck</td><td>P5731</td><td></td></tr><tr><td>PIPES</td><td>Merck</td><td>P1851</td><td></td></tr><tr><td>Pipet tips (0.5-10 uL) sterile&amp;nbsp;</td><td>Merck/Corning</td><td>AXYT300</td><td></td></tr><tr><td>Pipet tips (200 uL) sterile</td><td>Merck/Corning</td><td>AXYT200Y&amp;nbsp;</td><td></td></tr><tr><td>Pipet tips (1000 uL) sterile</td><td>Merck/Corning</td><td>AXYT1000BRS</td><td></td></tr><tr><td>Razor blade - single edged</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>Rocking platform shaker</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Spermine</td><td>Merck</td><td>85590</td><td></td></tr><tr><td>Spermidine&amp;nbsp;</td><td>Merck</td><td>S2626</td><td></td></tr><tr><td>Sorbitol</td><td>Merck</td><td>S8143</td><td></td></tr><tr><td>Tween-20</td><td>Merck</td><td>P9416</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>9002-93-1</td><td></td></tr><tr><td>Ultra pure Acrylamide</td><td>Invitrogen</td><td>15512023</td><td></td></tr><tr><td>Vacuum</td><td></td><td></td><td></td></tr></tbody></table>

three-dimensional immunolocalization in maize meiocytes

Three-Dimensional Immunolocalization In Maize Meiocytes

During meiotic prophase I, the physical configuration of chromosomes varies widely across developmental stages, fortunately these stage-specific configurations allow the proper identification of each of these classical meiotic stages, which are respectively known as leptotene (e.g. the &#8216;thin threads&#8217;), zygotene (e.g. the &#8216;coupled threads&#8217;), pachytene (e.g. the &#8216;thick threads&#8217;), diplotene (e.g. the &#8216;double threads&#8217;) and diakinesis (e.g. the &#8216;repulsion motion&#8217;). The visual analysis of several cytogenetic phenomena that occur during prophase I, such as recombination, pairing, and synapsis, requires highly reliable immunolocalization techniques. In contrast to traditional spreading and squashing techniques that suppress all subcellular and subnuclear information, the 3D conservation procedure described here allows the preservation of key meiotic proteins in their native subcellular location sites (cytoplasmic, nuclear, and subnuclear: euchromatin, heterochromatin, and nucleolar). Thus, this protocol describes how to efficiently fix meiotic anthers, dissect and release meiocytes from anthers, and perform simultaneous immunolocalization of three key proteins in maize male meiocytes.

Using this protocol, we can simultaneously observe the localization patterns of the three key meiotic proteins in maize (Figure 1). The meiotic protein ABSENCE OF FIRST DIVISION 1 (AFD1) is a homolog of the alpha-Kleisin REC8 subunit of the meiotic cohesin complex. The ASYNAPTIC 1 (ASY1) is a crucial axial element, while the ZYPPER1 (ZYP1) is the transversal element of the synaptonemal complex15. DAPI staining shows a clear nucleus and chromatin staining, allowing for...

This immunolocalization protocol in male maize meiocytes maintains the three-dimensional integrity of the cells during meiosis. It allows the dynamic tracking of the intracellular localization of up to three independent antibodies in preserved paraformaldehyde-fixed material, in addition to DAPI, which stains DNA.

During meiotic prophase I, the physical configuration of chromosomes varies widely across developmental stages, fortunately these stage-specific ...

Our research focuses on developing a 3D approach to study maize male meiocytes and visualize meotic proteins in single cells. Besides maize, this protocol can be applied to study meiocytes in rice, rye, barley, wheat. To begin, obtain maize plants at the V13 stage with influorescences still growing inside the stem.During the week of miotic anther fixation, gently press the enlarged stem at the bump while wearing gloves to check if it is soft enough to cut open. After preparing the required buffers, prepare a humid chamber by placing five to 10 humidified paper towels inside a plastic box. Remove excess water from the chamber.Using a razor blade, cut open the stem at the influorescence level to expose tassel branches and lateral branches. After dissecting and collecting the lateral branches, wrap them in the humid chamber towels while maintaining the chamber at room temperature. Using a stereo microscope, collect fresh anthers from florets into a 35 millimeter Petri dish containing two milliliters of one Xbuffer A or BA.Identify miotic stages based on size and color.After transferring the anthers to a new 35 millimeter Petri dish, add three milliliters of fixative solution to the anthers. Seal the Petri dish with parafilm to prevent formaldehyde evaporation. Place the 35 millimeter Petri dish on the larger Petri dish positioned on a rotary shaker, set to 40 to 60 RPM at room temperature for 45 minutes.Rinse the anthers with three milliliters of one XBA. After transferring the anthers to a small Petri dish, seal the Petri dish and place it on a rotary shaker at room temperature for 45 minutes with the speed set to 40 to 60 RPM. Using clean forceps, transfer the anthers to a small 35 millimeter Petri dish containing three milliliters of one XBA.Once the Petri dish is tightly sealed with parafilm, place the small Petri dish inside a larger Petri dish. After covering it with aluminum foil, store the sample at four degrees Celsius until use. On day one, prepare two milliliters of acrylamide solution by mixing equal volumes of acrylamide and two XBA.Degas the acrylamide solution under vacuum for five to 10 minutes before adding sodium sulfite and ammonium per sulfate. Aliquot 100 microliters of a one-to-one acrylamide and two XBA mixture into 1.5 milliliter tubes according to the number of slides prepared. Place two centimeter by two centimeter pieces of parafilm over a Petri dish and position them under a dissecting stereoscope.After trimming the pipette tip using scissors, pipette 50 microliters of BA solution with anthers onto the parafilm. Pipette 25 microliters of BA solution onto another area of the parafilm. After holding the anthers with forceps, using a scalpel, cut their tips.Once the meiocytes are squeezed out, use a pipette to transfer them into the 25 microliter BA drop and mix gently. Pipette 10 microliters of the meiocyte mixture onto a separate cover slip. Rapidly add five microliters of sodium sulfite and five microliters of ammonium per sulfate into a 1.5 milliliter tube containing the acrylamide and two XBA.Pipette five microliters of this mixture onto the cover slip and mix it with the meiocytes. Place a poly-lysine coated cover slip on top at a 45 degree angle. After allowing 30 to 50 minutes for full polymerization at room temperature, carefully separate the cover slips using a razor blade.Then, place them pad side up in a Petri dish. Rinse the cover slip with one milliliter of the 1XPBS supplemented with one millimolar ethylenediaminetetraacetic acid, or EDTA, and 1%Triton at room temperature for two hours. Prepare and combine primary antibodies from different host species to ensure distinct signals during detection.Add 50 microliters of the diluted primary antibody to the center of the cover slip containing the pad. Then, place the Petri dish in a humid chamber overnight. On day two, prepare 500 milliliters of wash buffer containing one XPBS, 0.1%Tween 20, and one millimolar EDTA.Using a pipette, remove the overnight solution. Wash the cover slip twice with one milliliter of wash buffer. After removing the buffer, again add one milliliter of wash buffer and incubate for one hour at room temperature.Repeat this washing step seven more times. After the final wash, cover the dish and leave it overnight in a humid chamber. Prepare the secondary antibodies by diluting them at a ratio of one to 50 in blocking buffer.After removing the wash solution, add 50 microliters of the diluted secondary antibody to the center of each pad. Then, place the Petri dish in a humid chamber for two hours at room temperature. After removing the solution from the well, wash four times with one milliliter of wash buffer for one hour each at room temperature.Wash the well with one milliliter of PBS for 10 minutes and repeat this process two more times. Add 500 microliters of DAPI, prepared at a final concentration of one microgram per milliliter to the well and incubate for 30 minutes at room temperature. After removing the prolonged gold from four degrees Celsius, thaw it to reach room temperature.Wash the slides with one milliliter of PBS for 30 minutes and repeat twice. After removing the final wash solution, add two drops of prolonged antifade in the middle of the pad. Once the cover slip is lifted from the well, mount it onto the labeled slides.Using a vacuum, remove excess glycerol from the edges of the cover slip and seal with two coats of nail polish. Observe the slides under a microscope using appropriate excitation wavelengths and filters to capture images. DAPI staining showed a clear nucleus and chromatin staining along with fainter native cytoplasmic staining of plastid and mitochondrial DNA.Absence of first division one or AFD1, asynaptic one or ASY1, and Zipper1 or ZYP1 localized on chromosomes and in the cytoplasm during preleptinema. In later stages, the signal concentrated on the chromosome, while substantial ZYP1 remained in the cytoplasm before complete synapsis. The meotic cohesin AFD1 formed a linear structure only after zygonema.Concomitantly with the formation of linear filaments of the synaptonemal complex. The ASY1 signal became comparatively faint after ZYP1 and AFD1 were loaded into linear structures. This protocol enables researchers to study protein co-localization and dynamics, native subcellular localization, 3D chromosomal architecture, meotic progression, and staging.The main challenge in this protocol is carefully selecting compatible combinations of primary and secondary antibodies. studies can built on this work by comparing male and female meiosis to investigate the molecular mechanism underlying athelchasmi.

three-dimensional-immunolocalization-in-maize-meiocytes

Research

JoVE Journal

Biology

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255 Views.  National Autonomous University of Mexico (UNAM). Our research focuses on developing a 3D approach to study maize male meiocytes and visualize meotic proteins in single cells. Besides maize, this protocol can be applied to study meiocytes in rice, rye, barley, wheat. To begin, obtain maize plants at the V13 stage with influorescences still growing inside the stem.During the week of miotic anther fixation, gently press the enlarged stem at the bump while wearing gloves to check if it is soft enough to cut open. After preparing the requ...

Three-Dimensional Immunolocalization In Maize Meiocytes

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